Previous work from this laboratory showed that it is possible to replace one nonmuscle myosin heavy chain (NMHC) in mice with another using homologous recombination. For example, we replaced NMHC II-B with NMHC II-A resulting in expression of NMHC II-A under control of the endogenous II-B promoter. Presently, we are studying the effects of replacing murine NMHC II-A with GFP-tagged human NMHC II-B. The targeting construct was generated by introducing the GFP-NMHC II-B-SV40 polyA casette into the first coding exon of the NMHC II-A genomic locus and the construct was electroporated into embryonic stem cells. As a control, mCherry-tagged human NMHC II-A-SV40 polyA was introduced into the same locus to replace endogenous NMHC II-A. Following both positive (neomycin) and negative (thymidine kinase) selection, candidate embryonic stem cell clones were analyzed. Surprisingly, in both cases, 23 out of 24 embryonic stem cell clones showed homologous recombination. We selected heterozygous clones for each construct and generated chimeric mice which showed evidence of germline transmission. We have generated small numbers of mice from F1 crosses of heterozygous mice. To date, no live homozygous mice have been born with NMHC II-B replacing II-A. However, unlike the NMHC II-A ablated mice which die before embryonic day (E)6.5, these homozygous mice survive to E9.5, suggesting that expressing nonmuscle myosin II-B in place of II-A can rescue early embryonic stages in development, such as gastrulation.